Here we describe a simple protocol for the outgrowth and differentiation of vasculogenic precursor cells (endothelial and vascular smooth muscle cells) from adult heart using Thymosin β4 to stimulate epicardial cell migration. The adult epicardium, unlike that of the embryo, has come to be regarded as a quiescent lineage, incapable of migration or differentiation. However, when given an appropriate stimulus, adult epicardium derived cells (EPDCs) proved capable of migration and differentiation into endothelial cells, smooth muscle cells and fibroblasts, the cell types known to derive from embryonic epicardium. We have identified Tβ4, an actin binding protein required for embryonic coronary vasculature formation, as a factor which can induce EPDC migration from adult heart. This straightforward protocol provides a means of enabling adult EPDC migration and a model system in which to study the ability of factors to influence the migration of vascular precursors and their differentiation. Models such as this will be invaluable for in vitro testing of factors prior to clinical trials for therapeutic angiogenesis.
In the developing embryo, the epicardium is the principal source of precursor cells for coronary vasculogenesis1. In the adult, the need to maintain a healthy coronary vasculature to meet the high demand for oxygen and nutrients for the myocardium is highlighted by the devastating consequences of coronary artery disease, which frequently results in extensive myocardial necrosis leading to cardiac failure.
Neovascularisation (new vessel formation) is an integral component of the cardiac remodelling process after myocardial infarction (MI). Numerous and dilated vessels appear in the border zone surrounding the infarct and as a result, coronary vasodilatory capacity resumes via an increase in blood flow
in the proximal region of the infarcted myocardium. However, neovascularisation is limited and insufficient to preserve viable myocardium.
A number of clinical and experimental trials of therapeutic angiogenesis, for example the administration of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), have been attempted to enhance neovasculogenesis and minimise cardiomyocyte loss after MI. However, phase II clinical trials for these factors, the VIVA2 and FIRST3 studies, respectively, were largely unsuccessful, due to a lack of knowledge of the precise mechanisms that underlie coronary angiogenesis.
Vascular regeneration includes adaptive vasculogenesis and arteriogenesis4 and the supply of endothelial and smooth muscle vascular precursors required for this process has been attributed, in part, to the peripheral circulation and bone marrow 5,6. However, a number of studies in humans reveal only very modest contributions of endothelial and smooth muscle cells from a bone marrow source7 and indeed it is still highly contentious as to whether adult multipotent progenitors from bone marrow participate in vessel formation4 or in myocardial regeneration (reviewed in8) following infarct.
The ability to mobilize endogenous progenitor cells from within the adult heart and to induce their differentiation into vascular cells capable of forming vessels offers tremendous potential for the treatment of human heart disease9. In this regard, the contribution of epicardium-derived endothelial and smooth muscle cells was hitherto believed to be confined entirely to embryonic epicardium and the key to unlocking the vasculogenic potential of adult epicardium has remained elusive.
Primary epicardial cells have been derived from fetal and early neonatal hearts10. Cultures from these stages assume an epithelial morphology and express several epicardial markers; explant cultures can be maintained for a number of days and passaged at least four times without alteration in epithelial morphology10. However, this protocol is limited in its application for use on embryonic and neonatal hearts (up to postnatal day 4). Trophic activity of the epicardium, in terms of its ability to stimulate fetal cardiomyocyte proliferation diminished rapidly between embryonic day 12 (E12) and P4 (ref. 10). Moreover, we determined that the number of EPDCs competent to migrate also diminished over a similar developmental time course11. We derived epicardial explants from E10.5, E12.5, E14.5 and E16.5 embryos and P1 neonates. Maximal outgrowth was observed at E10.5, a stage in development coincident with the formation of the epicardium. The ability of the embryonic heart to produce epicardial outgrowth diminished considerably by E12.5 and continued to do so such that by E16.5 the number of epicardial cells to migrate from the outgrowth was approximately 40% of that at E10.5 and further reduced to approximately 10% by P1. In untreated, adult explants there was virtually no detectable outgrowth (Fig. 1a) with only a few isolated cells observed in the culture dish (Fig. 1b). This is consistent with the adult epicardium residing in a quiescent state having lost migration, differentiation and signalling capacities during the latter half of gestation10.
We have identified the actin-binding protein, Thymosin β4 (Tβ4) as a factor required for all three stages of embryonic coronary vessel formation, namely vasculogenesis, angiogenesis and arteriogenesis11. In order to determine whether Tβ4 has the potential for therapeutic angiogenesis in adult heart, we treated adult heart explants with Tβ4. In contrast to untreated adult heart, Tβ4 stimulated extensive outgrowth of cells which, like embryonic cultures, displayed a characteristic epithelial morphology and were positive for the epicardial-specific transcription factor, epicardin (Fig. 1c,2a,c). As cells migrated away from the explant, they differentiated into a variety of discernable cell types (Fig.2b, d-f). Procollagen type I, SM alpha actin and Flk1 positive cells indicated the presence of fibroblasts, smooth muscle and endothelial cells, respectively (Fig. 2b, d-f). Smooth muscle cells and endothelial cells are definitive precursors for the coronary microvasculature and as such Tβ4-induced adult EPDCs represent a viable source of therapeutic vascular progenitors.